ABSTRACT
The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵* Co-first-authors
Firstly, we have updated the labels for Figures 3F, 5F, 5I, 6I, and 7F. Secondly, we have included the BioRender URL in the Acknowledgement section. Lastly, we have revised the Discussion section by incorporating several key pieces of important information, as listed below: 1. Single cell sequencing studies confirm that hepatoblasts expressing albumin are present at e11 and that undifferentiated endodermal cells (the precursors to hepatoblasts) are non-existent (Wang et al., 2020). 2. Could the ORC deletion lead to the immediate loss of hepatoblasts (despite having inherited ORC protein from the endodermal cells) causing undifferentiated endodermal cells to persist and proliferate much longer (without activating Cre) than in normal development? We consider this unlikely, but if true it will be very unexpected, both by suggesting that deletion of ORC immediately leads to the death of the hepatoblasts (despite a healthy reserve of inherited ORC protein) and by suggesting that there is a novel feedback mechanism from the death/ORC depletion of hepatoblasts leading to the persistence and proliferation of undifferentiated endodermal cells. 3. We have also shown that loss of ORC subunits in cancer cell lines causes significant changes in gene expression due to the role of ORC subunits in epigenetic regulation(Su et al., 2024), so another explanation of the more severe phenotype in females could be that the epigenetic effects of ORC subunits are more important in female compared to male livers. 4. It is also worth noting that in cancer cells in culture, we are seeing a near normal level of loading of MCM2-7 on chromatin when Orc1, Orc2 or Orc5 genes are deleted, and that 60% of the origins of replication remain at the same sites as in WT cells (Shibata et al., 2024). Although it is impossible to rule out that a very small amount of ORC protein somehow persists in the hepatocytes (or the cancer cell lines) with these mutations and that this is sufficient to facilitate loading of enough MCM2-7 to support DNA synthesis in vivo or in vitro, this is becoming progressively unlikely.
